The specific aims of this research program are to study the regulation of albumin and alpha-fetoprotein (AFP) expression in mouse hepatoma x rat fibroblast cell hybrids. Albumin and AFP synthesis, in the mouse hepatoma parent cell, are repressed when somatic cell hybrids are formed by fusion with rat fibroblast cells. Experiments will be done to determine whether this repression involves inhibition of transcription; a defect in the processing of nuclear RNA transcripts, or the transport of the mature mRNA to the cytoplasm. Major emphasis will be placed on studying the chromatin structure of the active and repressed genes in the parent and hybrid cells. DNase 1, restriction endonuclease and micrococcal nuclease digestion analyses will be done to determine the nucleosomal organization of albumin and AFP chromatin in the parent and hybrid cells. Using specific probes derived from cDNA and genomic clones, experiments will be done to determine whether the chromatin of the repressed genes shows a supernucleosomal structure and 5'-end hypersensitivity to controlled DNase I digestion. Studies will also be done to determine whether gene activation is associated with the loss of supernucleosomal organization within the genes and their flanking regions. In our recent studies we have shown that repression of the albumin and AFP genes in the hybrid cells is associated with methylation of DNA sequences at the 5'-end and flanking regions. In our ongoing research we are attempting to localize specific sites of methylation in this regulatory event. Attempts will be made to activate albumin and AFP synthesis in the hybrids by treatment with glucocorticoids for albumin activation and hepatocarcinogens for AFP activation. Mechanisms of albumin and AFP gene activation will also be studied in hybrids set up specifically for this regulatory event. Using recently constructed albumin and AFP minigenes in the bovine papilloma vector, we propose to determine if regulatory proteins can be isolated from these corresponding minichromosomes after they have been transferred into the appropriate somatic cell hybrids. (P)